Succinylacetone (SA) is a potent specific inhibitor of heme biosynthesis binding irreversibly to delta-aminolevulinic acid dehydrase, the second enzyme of the pathway. When murine erythroleukemia (MEL) cells were grown in defined medium containing SA, cellullar heme concentrations decreased markedly and growth was inhibited. Growth inhibition was partially reversed when hematin was added with SA on day 0, but no reversal of growth inhibition was seen when hematin was added on day 4. The decrease of cell heme concentration was accompanied by a decrease of protein synthesis, which was partially reversed by addition of hematin to the medium. When hematin was added to the culture medium of cells which had been depleted of heme by the effect of SA, the intracellular heme levels returned to normal. Growth inhibition produced by SA was also reversed by adding hematin in the presence of SA. When heme-depleted cells were resuspended in fresh medium without SA, but containing hematin, normal levels of heme were attained within 2 hrs. The rate of heme uptake in cells incubated at both 20 degrees and 37 degrees was essentially the same. Both heme-depleted and cells with normal levels of heme showed uptake of hematin. SA inhibited L1210 tumor cell growth in vitro and increased the survival time of rats infected with Walker 256 ascites tumors.